U.S. patent application number 13/884595 was filed with the patent office on 2013-09-05 for method and device for high-throughput solution exchange for cell and particle suspension.
The applicant listed for this patent is Dino Di Carlo, Daniel R. Gossett, Henry T.K. Tse. Invention is credited to Dino Di Carlo, Daniel R. Gossett, Henry T.K. Tse.
Application Number | 20130228530 13/884595 |
Document ID | / |
Family ID | 46084582 |
Filed Date | 2013-09-05 |
United States Patent
Application |
20130228530 |
Kind Code |
A1 |
Di Carlo; Dino ; et
al. |
September 5, 2013 |
METHOD AND DEVICE FOR HIGH-THROUGHPUT SOLUTION EXCHANGE FOR CELL
AND PARTICLE SUSPENSION
Abstract
A method of exchanging fluids with suspended particles includes
providing a microfluidic device with a first inlet channel
operatively coupled to a source of particles and a second inlet
channel operatively coupled to an exchange fluid. A transfer
channel is connected at a proximal end to the first inlet channel
and the second inlet channel. First and second outlet channels are
connected to a distal end of the transfer channel. The source of
particles is flowed at a first flow rate into the first inlet
channel while the exchange fluid is flowed at a second flow rate
into the second inlet channel wherein the ratio of the second flow
rate to the first flow rate is at least 1.5. Particles are
collected in one of the first and second outlet channels while
fluid substantially free of particles is collected in the other of
the first and second outlet channels.
Inventors: |
Di Carlo; Dino; (Los
Angeles, CA) ; Gossett; Daniel R.; (Los Angeles,
CA) ; Tse; Henry T.K.; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Di Carlo; Dino
Gossett; Daniel R.
Tse; Henry T.K. |
Los Angeles
Los Angeles
San Francisco |
CA
CA
CA |
US
US
US |
|
|
Family ID: |
46084582 |
Appl. No.: |
13/884595 |
Filed: |
November 14, 2011 |
PCT Filed: |
November 14, 2011 |
PCT NO: |
PCT/US11/60536 |
371 Date: |
May 9, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61415067 |
Nov 18, 2010 |
|
|
|
Current U.S.
Class: |
210/767 ;
210/511; 210/85 |
Current CPC
Class: |
B01L 2300/0816 20130101;
B01L 2400/0487 20130101; B01L 2400/084 20130101; B01L 3/502746
20130101; B01L 3/502776 20130101; B01D 12/00 20130101; B01L
3/502761 20130101; G01N 2015/149 20130101; B01L 2200/0652
20130101 |
Class at
Publication: |
210/767 ;
210/511; 210/85 |
International
Class: |
B01D 12/00 20060101
B01D012/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with Government support under Grant
No. W81XWH-10-1-0519, awarded by the U.S. Army, Medical Research
and Materiel Command and Grant No. N66001-11-1-4125 awarded by the
U.S. Navy, Office of Navy Research. The Government has certain
rights in this invention.
Claims
1. A microfluidic system for solution exchange comprising: a first
inlet channel operatively coupled to a source of particles
suspended in a fluid; a second inlet channel operatively coupled to
an exchange fluid; a transfer channel having a proximal end and a
distal end, the proximal end of the transfer channel connected to
the first inlet channel and the second inlet channel; at least one
outlet channel connected to a distal end of the transfer channel;
and; a first pump configured to pump the source of particles
suspended in a fluid at a first flow rate and a second pump
configured to pump the exchange fluid at a second flow rate wherein
the ratio of the second flow rate to the first flow rate is at
least 1.5.
2. The microfluidic system of claim 1, wherein the first inlet
channel comprises a focusing region.
3. The microfluidic system of claim 1, wherein the focusing region
comprises a plurality of curves.
4. The microfluidic system of claim 1, wherein a width of the
transfer channel is substantially equal to the sum of the width of
the first inlet channel and the second inlet channel.
5. The microfluidic system of claim 1, wherein the particles
comprise cells.
6. The microfluidic system of claim 1, wherein the length of the
transfer channel is at least 1 cm.
7. The microfluidic system of claim 1, wherein at least 1,000
particles per second enter the transfer channel.
8. The microfluidic system of claim 1, further comprising one or
more additional outlet channels.
9. The microfluidic system of claim 8, wherein the at least one
outlet channel comprises a collection channel or a waste
channel.
10. The microfluidic system of claim 1, further comprising an
analyzer downstream of the at least one outlet channel.
11. The microfluidic system of claim 10, wherein the analyzer
comprises a flow cytometer.
12. The microfluidic system of claim 10, wherein the analyzer
comprises a fluorescent-activated cell sorter (FACS).
13. The microfluidic system of claim 10, wherein the analyzer
comprises an imager.
14. A microfluidic system for solution exchange comprising: a first
inlet channel operatively coupled to a source of particles
suspended in a fluid; a second inlet channel operatively coupled to
an exchange fluid; a first transfer channel having a proximal end
and a distal end, the proximal end of the first transfer channel
connected to the first inlet channel and the second inlet channel;
at least one outlet channel connected to a distal end of the first
transfer channel; and; a first pump configured to pump the source
of particles suspended in a fluid at a first flow rate and a second
pump configured to pump the exchange fluid at a second flow rate
wherein the ratio of the second flow rate to the first flow rate is
at least 1.5; a third inlet channel operatively coupled to the at
least one outlet channel; a second transfer channel having a
proximal end and a distal end, the proximal end of the second
transfer channel connected to the third inlet channel and the at
least one outlet channel; a third pump configured to pump a second
exchange fluid into the third inlet channel; and at least one
outlet channel connected to a distal end of the second transfer
channel.
15. The microfluidic system of claim 14, wherein the third pump is
configured to pump the second exchange fluid at a flow rate that is
at least 1.5 times the flow rate of fluid through the at least one
outlet channel connected to a distal end of the first transfer
channel.
16. The microfluidic system of claim 14, wherein the second
exchange fluid is different from the first exchange fluid.
17. The microfluidic system of claim 14, wherein the particles
comprise cells.
18. The microfluidic system of claim 14, wherein the length of the
transfer channel is at least 1 cm.
19. The microfluidic system of claim 14, wherein at least 1,000
particles per second enter the first and second transfer
channels.
20. The microfluidic system of claim 14, further comprising one or
more additional outlet channels.
21. The microfluidic system of claim 20, wherein the at least one
outlet channel comprises a collection channel or a waste
channel.
22. The microfluidic system of claim 14, further comprising an
analyzer downstream of the at least one outlet channel.
23. The microfluidic system of claim 22, wherein the analyzer
comprises a flow cytometer.
24. The microfluidic system of claim 22, wherein the analyzer
comprises a fluorescent-activated cell sorter (FACS).
25. The microfluidic system of claim 22, wherein the analyzer
comprises an imager.
26. A method of exchanging fluids with suspended particles
comprising: providing a microfluidic device comprising a first
inlet channel operatively coupled to a source of particles
suspended in a fluid; a second inlet channel operatively coupled to
an exchange fluid; a transfer channel having a proximal end and a
distal end, the proximal end of the transfer channel connected to
the first inlet channel and the second inlet channel; and first and
second outlet channels connected to a distal end of the transfer
channel; flowing the source of particles suspended in a fluid at a
first flow rate into the first inlet channel; flowing the exchange
fluid at a second flow rate into the second inlet channel wherein
the ratio of the second flow rate to the first flow rate is at
least 1.5; collecting particles in one of the first and second
outlet channels; and collecting fluid substantially free of
particles in the other of the first and second outlet channels.
27. The method of claim 26, wherein the suspended particles
comprise cells.
28. The method of claim 27, wherein the suspended particles
comprise cells and beads.
29. The method of claim 26, wherein the exchange fluid comprises a
wash fluid.
30. The method of claim 26, wherein the exchange fluid comprises a
dye.
31. The method of claim 26, further comprising analyzing the
collected particles with an analyzer selected from the group
consisting of a flow cytometer, fluorescent-activated cell sorter
(FACS), and imager.
32. The method of claim 26, wherein the microfluidic device further
comprises a third inlet channel operatively coupled to the outlet
channel collecting particles and a second transfer channel having a
proximal end and a distal end, the proximal end of the second
transfer channel connected to the third inlet channel and the
outlet channel collecting particles and wherein third and fourth
outlet channels connect to a distal end of the second transfer
channel; flowing a second exchange fluid into the third inlet
channel; collecting particles in one of the third and fourth outlet
channels; and collecting fluid substantially free of particles in
the other of the third and fourth outlet channels.
33. The method of claim 32, wherein the flow rate of the second
exchange fluid is at least 1.5 times the flow rate of fluid through
particle containing outlet channel connected to a distal end of the
first transfer channel.
Description
RELATED APPLICATION
[0001] This Application claims priority to U.S. Provisional Patent
Application No. 61/415,067 filed on Nov. 18, 2010. Priority is
claimed pursuant to 35 U.S.C. .sctn.119. The above-noted Patent
Application is incorporated by reference as if set forth fully
herein.
FIELD OF THE INVENTION
[0003] The field of the invention generally relates to microfluidic
devices. More particularly, the field of the invention relates to
microfluidic devices used in solution exchange applications for
cell and particle suspensions
BACKGROUND
[0004] The current standard technique to transfer particles or
cells from one solution to another at the macroscale level involves
centrifugation and re-suspension. This is a manual labor and time
intensive process that is not easily miniaturized or integrated due
to the bulk of the centrifuge machine and manual pipetting steps
required. Centrifugation and pipetting steps are, of course, labor
and time intensive processing steps. Attempts have been made to
miniaturize this capability using microstructures to divert cells
while not diverting the fluid component. For example, Morton et al.
discloses an asymmetric post array used in pressure-driven
microfluidic flow to move particles of interest across multiple,
independent chemical streams. See Morton, K. J. et al., Crossing
microfluidic streamlines to lyse, label and wash cells, Lab Chip 8,
1448-1453 (2008).
[0005] Others have used dielectrophoresis (DEP) to transfer
particles electrically. For example, Tronay et al. have used
activated DEP electrodes in a microfluidic device where particles
can be continuously functionalized in flow. The device uses a
particle exchanger which allows for particles to be taken from one
medium and exposed to some reagent while minimizing mixing of the
two liquids. In the exchanger, two liquids are brought in contact
and particles are pushed from one to the other by the application
of a dielectrophoretic force. See Tornay, R. et al.,
Dielectrophoresis-based particle exchanger for the manipulation and
surface functionalization of particles, Lab Chip 8, 267-273 (2008).
Still others have used acoustic manipulation of suspended
particles, in which particles in a laminar flow microchannel are
continuously translated from one medium to another with virtually
no mixing. See Petersson, F. et al., Carrier Medium Exchange
through Ultrasonic Particle Switching in Microfluidic Channels,
Anal. Chem. 77, 1216-1221 (2005). Yet another approach uses
hydrodynamic filtration in which the virtual width of flow in a
microchannel determines the size of filtered cells/particles. See
Yamada M. et al., Millisecond treatment of cells using microfluidic
devices via two-step carrier medium exchange, Lab Chip, 8, 772-778
(2008).
[0006] While some microfluidic-based sorting devices have been
proposed for solution exchange, there are concerns about device
complexity, and the speed of operation. In many cases, the speed of
exchange is rather slow and cannot be integrated, for example, with
additional downstream processing applications such as cytometry.
Microfluidic-based solution exchange systems should have
high-throughput, be easy to multiplex, should be able to position
particles or cells for possible downstream interrogation, and
should have fast transfer. The prior techniques do not satisfy all
of these criteria.
SUMMARY
[0007] In one embodiment, a microfluidic system for solution
exchange includes a first inlet channel operatively coupled to a
source of particles suspended in a fluid and a second inlet channel
operatively coupled to an exchange fluid. The system includes a
transfer channel having a proximal end and a distal end, the
proximal end of the transfer channel connected to the first inlet
channel and the second inlet channel. At least one outlet channel
is connected to a distal end of the transfer channel. A first pump
is configured to pump the source of particles suspended in a fluid
at a first flow rate and a second pump is configured to pump the
exchange fluid at a second flow rate wherein the ratio of the
second flow rate to the first flow rate is at least 1.5.
[0008] In another embodiment, a microfluidic system for solution
exchange includes a first inlet channel operatively coupled to a
source of particles suspended in a fluid and a second inlet channel
operatively coupled to an exchange fluid. The system includes a
first transfer channel having a proximal end and a distal end, the
proximal end of the first transfer channel connected to the first
inlet channel and the second inlet channel. At least one outlet
channel is connected to a distal end of the first transfer channel.
A first pump is configured to pump the source of particles
suspended in a fluid at a first flow rate and a second pump
configured to pump the exchange fluid at a second flow rate wherein
the ratio of the second flow rate to the first flow rate is at
least 1.5. The system includes a third inlet channel operatively
coupled to the at least one outlet channel and a second transfer
channel having a proximal end and a distal end, the proximal end of
the second transfer channel connected to the third inlet channel
and the at least one outlet channel. A third pump is configured to
pump a second exchange fluid into the third inlet channel. At least
one outlet channel is connected to a distal end of the second
transfer channel.
[0009] In another embodiment, a method of exchanging fluids with
suspended particles includes providing a microfluidic device
comprising a first inlet channel operatively coupled to a source of
particles suspended in a fluid and a second inlet channel
operatively coupled to an exchange fluid, a transfer channel having
a proximal end and a distal end, the proximal end of the transfer
channel connected to the first inlet channel and the second inlet
channel, and first and second outlet channels connected to a distal
end of the transfer channel. The source of particles suspended in a
fluid is flowed at a first flow rate into the first inlet channel.
The exchange fluid is flowed at a second flow rate into the second
inlet channel wherein the ratio of the second flow rate to the
first flow rate is at least 1.5. Particles are collected in one of
the first and second outlet channels. Fluid substantially free of
particles is collected in the other of the first and second outlet
channels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 illustrates a microfluidic system according to one
embodiment.
[0011] FIGS. 2A-2D are high speed photographic images taken of a
transfer channel 36 that includes co-flow of a fluid containing a
particle 14 (a 19 .mu.m microsphere) as well as a dye fluid
(darker, lower fluid).
[0012] FIG. 3A illustrates a top view of an alternative embodiment
of a microfluidic system that incorporates downstream analysis.
[0013] FIG. 3B illustrates a side cross-sectional view of the
embodiment of FIG. 3A taken along the line B-B'.
[0014] FIG. 3C illustrates a side cross-sectional view of the
embodiment of FIG. 3A taken along the line C-C'.
[0015] FIG. 3D illustrates a side cross-sectional view of the
embodiment of FIG. 3A taken along the line D-D'.
[0016] FIG. 4 illustrates a microfluidic system according to
another embodiment.
[0017] FIG. 5 illustrates a microfluidic system according to
another embodiment that is can be used in histological staining
applications.
[0018] FIG. 6 illustrates a microfluidic system according to
another embodiment.
[0019] FIG. 7 illustrates a microfluidic system according to
another embodiment.
[0020] FIG. 8A illustrates a microfluidic system according to
another embodiment.
[0021] FIG. 8B illustrates a cross-sectional view of the transfer
channel taken along the line B-B' of FIG. 8A.
DETAILED DESCRIPTION OF THE ILLUSTRATED EMBODIMENTS
[0022] FIG. 1 illustrates a microfluidic system 10 for solution
exchange according to one embodiment. The microfluidic system 10
includes a first inlet channel 12 that is operatively coupled to
source of particles 14 suspended in a fluid 16. The term
"particle," as used herein, is meant to refer broadly to objects
capable of being suspended in fluid 16. Exemplary particles include
such things as beads, cells, vesicles, micelles, and the like. The
particles 14 and the fluid 16 are flowed into the first inlet
channel 12 using a pump 18 such as a syringe pump as illustrated in
FIG. 1 although other pumping devices may be used. The pump 18
interfaces with first inlet channel 12 at an inlet 20. The inlet 20
may include optional filters 22 downstream thereof which may be
structures dimensioned (e.g., posts, fins, or the like) to trap
large debris and aggregates while allowing passage of particles
14.
[0023] Still referring to FIG. 1, the first inlet channel 12 may
have a width within the range of about 50 .mu.m to about 100 .mu.m
and a height within the range of about 20 .mu.m to about 30 .mu.m
although other dimensions may be used. The first inlet channel 12
may include an optional focusing region 24. The focusing region 24
may be used to focus the particles 14 laterally and/or
longitudinally within the first inlet channel 12. In one aspect,
the focusing region 24 includes a plurality of curves 26 which may
be symmetrically or asymmetrically shaped to promote focusing.
Arrows A in FIG. 1 illustrate inertial forces within the curves 26
that focus the particles 14. Examples of such focusing structures
may be found, for example, in the publication by DiCarlo et al.,
Continuous inertial focusing, ordering, and separation of particles
in microchannels, Proc. Natl. Acad. Sci. 104(48): 18892-18897
(2007), which is incorporated by reference herein. Another type of
focusing region 24 may be found in U.S. patent application Ser. No.
13/230,559, which is also incorporated by reference herein. In
still another option, the first inlet channel 12 may be lengthened
because a longer length microchannel tends to focus particles 14
therein.
[0024] Still referring to FIG. 1, the microfluidic system 10
includes a second inlet channel 28 that is operatively coupled to
an exchange fluid 30. The exchange fluid 30 may include any number
of fluids. For example, the exchange fluid 30 may include a wash
such as phosphate buffered saline (PBS). The exchange fluid 30 may
also include a reagent or the like therein. For example, the
exchange fluid 30 may include a dye or label therein. The exchange
fluid 30 is flowed into the second inlet channel 28 using a pump 32
such as a syringe pump as illustrated in FIG. 1 although other
pumping devices may be used. The pump 32 interfaces with second
inlet channel 28 at an inlet 34. The inlet 34 may include optional
filters 22 located downstream thereof the same as or similar to
those described in the context of the first inlet channel 12. The
second inlet channel 28 may have a width within the range of about
50 .mu.m to about 100 .mu.m and a height within the range of about
20 .mu.m to about 30 .mu.m although other dimensions may be used.
The length of the second inlet channel 28 may vary but is typically
a few millimeters (e.g., 2 mm)
[0025] Still referring to FIG. 1, both the first inlet channel 12
and the second inlet channel 28 are connected to a downstream
transfer channel 36. The transfer channel 36 has a proximal end 38
and a distal end 40 with the first inlet channel 12 and the second
inlet channel 28 intersecting with the proximal end 38 of the
transfer channel 36. Both the first inlet channel 12 and the second
inlet channel 28 are angled relative to the transfer channel 36.
Preferably, the first inlet channel 12 and the second inlet channel
28 are symmetrically angled relative to the transfer channel 36.
For example, both the first inlet channel 12 and the second inlet
channel 28 may be angled (.alpha.) approximately at or less than
60.degree. relative to each other. The transfer channel 36
typically has a height that is the same as the height of the first
inlet channel 12 and the second inlet channel 28 however, in
multiplexed embodiments it is possible to have multi-planar
structures in which case the height does not have to be equal to
that of the first inlet channel 12 and the second inlet channel 28.
The width of the transfer channel 36 is generally roughly equal to
the summation of the respective widths of the first inlet channel
12 and the second inlet channel 28. For example, widths of the
transfer channel 36 typically fall within the range of about 75
.mu.m to about 100 .mu.m. Generally, the cross-sectional aspect
ratio of the height to width (H:W) is less than 1 and preferably
less than 2/3 (e.g., 0.5). The length of the transfer channel 36 is
typically greater than or equal to about 1 cm although other
lengths may be used.
[0026] In one aspect of the invention, the flow rate at which the
particles 14 suspended in the fluid 16 are flowed into the first
inlet channel 12 is different than the flow rate at which the
exchange fluid 30 is flowed into the second inlet channel 28. In
this regard, the exchange fluid 30 should be flowed into the second
inlet channel 28 at a flow rate that exceeds the flow rate of the
particles 14 suspended in fluid 16. More particularly, it has been
found that the ratio of the flow rate of the exchange fluid 30 to
the flow rate of the particles 14 suspended in fluid 16 should be
at least about 1.5. In another aspect of the invention, the ratio
is within the range of about 1.5 to about 2.0. Typically, the flow
rate for the particles 14 suspended in fluid 16 may be in the range
of about 50 .mu.l/min. to about 80 .mu.l/min. The flow rate for the
exchange fluid 30 may be in the range of about 90 .mu.l/min. to
about 120 .mu.l/min although flow rates outside this range may also
be used. Generally, the microfluidic device 10 should be
constructed such that a Particle Reynolds Number (R.sub.p) is
between the range of about 0.25 to about 1.87. R.sub.p is defined
as follows:
R.sub.p=.rho.U a.sup.2/.mu.H (Eq. 1)
[0027] where .rho. is the density, U is the maximum channel
velocity, a is the particle diameter, .mu. is the viscosity, and H
is the channel height. Moreover, the Reynolds number of the fluid
flowing through the first inlet channel 12 and the second inlet
channel 28 should be less than about 2,000 so as to maintain
laminar and not turbulent flow.
[0028] The flow rate is high such that an interface 42 is formed
between the fluid 16 containing the particles 14 and the exchange
fluid 30. This interface 42 is advantageously maintained along
substantially the entire length of the transfer channel 36. In this
regard, a co-flow state is maintained between the exchange fluid 30
and the fluid 16 containing the particles 14 which is maintained
throughout the length of the transfer channel 36. Maintenance of
this interface 42 and the establishment of the co-flow state means
that there is very little diffusion across this interface. The
Peclet number (ratio of convection to diffusion) is typically high
(e.g., .about.160,000) such that diffusion across the interface 42
is negligible. Of course, the invention is not limited to a
particular Peclet number.
[0029] Still referring to FIG. 1, the microfluidic system 10
includes a plurality of outlet channels 44, 46. While two such
outlet channels are illustrated in FIG. 1, in other embodiments
there may be more outlet channels. The outlet channels 44, 46 have
dimensions to alter their respective fluidic resistance values. In
some embodiments, the outlet channels 44, 46 may have the same
height as the upstream transfer channel 36 but different widths so
as to adjust the rate of fluid volume which passes through each
outlet channel 44, 46. In the embodiment of FIG. 1, the particles
14 first enter the transfer channel 36 containing with carrier
fluid 16. The particles 14 feel inertial lift forces indicated by
arrows A that push the particles 14 toward an equilibrium position
(X.sub.eq) that generally lies within the center region of the
transfer channel 36 (centerline is shown by dashed line 48).
However, because the flow rate of the exchange fluid 30 is higher
than the flow rate of the fluid 16 with suspended particles 14,
more than half of the transfer channel 36 is filled with exchange
fluid 30. This can be seen in cross-sectional view of the transfer
channel 36 in FIG. 1 where the level of exchange fluid 30 is above
the centerline 48.
[0030] As stated above, the equilibrium position (X.sub.eq) in the
transfer channel 36 lies within the exchange fluid 30. Particles 14
are thus pushed by inertial lift forces from the suspension fluid
16 of the co-flow and into the exchange fluid 30 of the co-flow.
The particles 14 after reaching the equilibrium position (X.sub.eq)
are completely within the exchange fluid 30. The particles 14 are
thus exchanged from the original suspension fluid 16 to the
exchange fluid 30. The particles 14 continue to travel downstream
toward the junction of the plurality of outlet channels 44, 46. In
the embodiment of FIG. 1, the particles 14 exit the transfer
channel 36 and enter outlet channel 44. The other outlet channel 46
which is angled relative to the transfer channel 36 collects the
original fluid 16 used for suspension of the particles 14 along
with some of the exchange fluid 30. Substantially all or all of the
particles 14 however will be collected in outlet channel 44. In
this embodiment, outlet channel 44 collects the particles 14 and
exchange fluid 30 while the other outlet channel 46 contains no
particles 14 but a combination of the original fluid 16 plus some
collection fluid 30. In this regard, outlet channel 46 contains
fluid that is substantially free of particles 14. The ratio of
fluid resistance of the two outlet channels 44, 46 is adjusted
either through the geometric construction of the channels 44, 46 or
through some sort of application of pressure (either positive or
negative). For example, in the microfluidic system 10 of FIG. 1 it
has been found that a ratio of fluid resistance of around 2.6
(resistance of outlet channel 46: outlet channel 44) although this
number varies depending on the orientation of the outlets 44, 46 as
well as the total number of outlets.
[0031] FIGS. 2A-2D are high speed photographic images taken of a
transfer channel 36 that includes co-flow of a fluid containing a
particle 14 (a 19 .mu.m microsphere) as well as a dye fluid
(darker, lower fluid). FIG. 2A illustrates the particle 14 at the
initial time (t=0.00 ms) whereby the particle is entering the
transfer channel 36 from the first inlet channel 12. FIG. 2B
illustrates the particle 14 being subject to the inertial lift
forces (F.sub.L) at a time of 0.51 ms. The particle 14 is beginning
to move toward the equilibrium position (X.sub.eq). FIG. 2C
illustrates the same particle 14 at a time of 1.50 ms whereby the
particle 14 is continuing the migration into the dye. FIG. 2D
illustrates the particle 14 at the equilibrium position (X.sub.eq)
which completely lies within the dye solution.
[0032] The outlet channels 44, 46 are coupled to respective outlet
chambers 50, 52 in FIG. 1 where the particles 14 and fluids are
then contained (e.g., waste in chamber 52). Alternatively, one or
more of the outlet channels 44, 46 may interface with additional
microfluidics or devices for subsequent processing and/or analysis.
For example, FIG. 3 illustrates one such embodiment of a
microfluidic system 10 that incorporates downstream analysis. FIGS.
3A-3D illustrate a similar embodiment as that illustrated in FIG. 1
with one difference being the presence of three (3) outlet channels
54, 56, 58. In this embodiment, as best seen in FIG. 3A, the
particles 14 are cells and the fluid 16 contains fluorescent probes
60. The first inlet channel 12 is flowed at a flow rate of 60
.mu.L/minute while the second inlet channel 28 is flowed with a
wash solution 30 such as PBS at a rate of 90 .mu.L/minute (other
flow rates could also be used). As seen in FIG. 3B, the cells 14
are initially within the fluid 16 that contains the fluorescent
probes 60. The cells 14 migrate out of the probe-containing stream
of the co-flow and into the PBS exchange solution 30 within the
transfer channel 36. This is seen in FIG. 3C which illustrates a
cross-sectional view of the transfer channel 36. At the outlet
junction, cells 14 contained within the PBS exchange solution 30
flow into outlet channel 56 while outlet channels 54, 58 collect,
respectively, probe solution 16 and PBS exchange solution 30 as
seen in FIG. 3D. The outlet channel 56 continues on to analyzer 62.
The analyzer 62 may include any number of analysis devices such as
a flow cytometer, fluorescent-activated cell sorter (FACS), and
imager. For example, in the embodiment of FIGS. 3A-3D, the analyzer
62 may comprise a fluorescent detection device that detects
fluorescent cells 14 through laser interrogation.
[0033] One benefit of the microfluidic system 10 described herein
is that cells 14 can quickly be interrogated. In the embodiment of
FIGS. 3A-3D, for example, the fluorescent probe 60 may have weak
binding affinity to the cell 14 and after binding quickly
disassociates with the underlying cell 14. With this microfluidic
system 10, the quick exchange of the solution as well as the rapid
downstream analysis permits interrogation before the fluorescent
probe 60 disassociates with the cell 14. Cells 14 are flow directly
into the analyzer 62 without any time-consuming wash steps being
needed. Further, the signal-to-noise ratio is improved by
eliminating background fluorophores via outlet channel 54. While
flow cytometry can usually discriminate between free and bound
fluorescent probes, the study of low affinity interactions requires
higher concentrations of free probes which hinder the accuracy of
the flow cytometer in this task. With this microfluidic system 10
probe-bound objects can be transferred to a new solution with low
background, and with the implementation of a fluorescence detection
system, immediately record fluorescence measurements.
[0034] The microfluidic system 10 may be used for many different
applications. One primary application of the rapid solution
exchange approach disclosed herein is sample preparation. The
primary tasks in sample preparation include labeling of cells with
targets such as antibodies which are then washed to remove un-bound
antibodies. For example, cells could be incubated with antibody
then run through the microfluidic system 10 which then transfers
labeled cells to a clean solution. These cells could then be
analyzed using an inline fluorescence detection system such as that
illustrated in FIG. 3A whereby fluorescence measurements can be
immediately obtained. Alternatively, the cells could be analyzed
offline. Another application of the device is in the sample
preparation involving blood. When working with blood, it is
typically important to remove the red blood cells (RBCs). RBC
removal is accomplished by hypotonic lysis. One use of the
microfluidic system 10 is mixing whole blood containing RBCs with
lysis buffer and fluorescent label targeted to white blood cells.
FIG. 4 illustrates an example of the microfluidic system 10 being
used in this manner.
[0035] As seen in FIG. 4, the solution contained lysed RBCs (i.e.,
ghosts), hemoglobin, and nucleic acid stains (targeting white blood
cells and incubated for 10 minutes) were injected into the first
inlet channel 12 at a rate of 60 .mu.L/min. A PBS solution was
flowed in the second inlet channel 28 at a rate of 120 .mu.L/min.
The RBC ghosts and hemoglobin establish one part of the co-flow
within the transfer channel 36 while the PBS solution establishes
the remaining portion (lower portion in FIG. 4) of the co-flow in
the transfer channel 36. The white blood cells 14, with stained
nuclei, then migrate due to inertial forces into the PBS solution.
The entrained white blood cells then exit via a first outlet 44
while the RBC debris and hemoglobin leave via second outlet 46.
Table 1 below illustrates the measured relative background
fluorescence per 100 .mu.m.sup.2.
TABLE-US-00001 TABLE 1 Channel Relative background fluorescence per
100 .mu.m.sup.2 Inlet 1.00 Reject (RBCs) 0.89 Collect (WBCs)
0.30
[0036] The microfluidic system 10 can also be used in histological
staining applications as illustrated in FIG. 5. Here a microfluidic
system 10 similar to that disclosed in FIG. 1 is used in connection
with the staining of cells 14. In this example, MCF7 cells were
mixed with a stain (Methylene Blue) and flowed into the first inlet
channel at a rate of 60 .mu.L/min. A PBS solution was flowed in the
second inlet channel 28 at a rate of 110 .mu.L/min. The stained
cells migrated to the "clean" PBS portion of the co-flow while the
stain remained in the remaining portion of the co-flow. The
entrained stained cells then exit via a first outlet 44 while the
stain and other non-cell material leave via second outlet 46. In
this example, the first outlet 44 captures 96% of the cells.
[0037] FIG. 6 illustrates still another application of the
microfluidic system 10. In this embodiment, particles that are
cells 14 are mixed with beads 15 with binding affinity to cells 14
(e.g., functionalized beads or magnetic beads) some of which coat
the exterior of the cells 14. The cells 14 along with the beads 15
are flowed into the first inlet channel 12. A PBS or other "clean"
solution was flowed in the second inlet channel 28. The cells 14
having beads 15 bound thereto migrated to the "clean" PBS exchange
solution 30 portion of the co-flow while the free beads 15 remain
in the remaining portion of the co-flow. The bead-laden cells 14
then exit via a first outlet 44 while the free beads 15 leave via
second outlet 46.
[0038] Another application of the microfluidic device 2 is that
particles 14 may be used in conjunction with solution exchange to
sequester or elute targets of interest. For example, particles 14
with sizes such that they are subject the same to inertial forces
(e.g., the size of cells) may have surfaces functionalized that
bind to molecules of interest (e.g., targets). The particles 14 can
then be used to bind with the target species and collected while
the unwanted molecules can be removed via a waste stream. The
reverse could also be employed. For example, previously washed
particles 14 having bound targets thereon could be brought into a
solution where the molecules elute from the particles 14. The
particles 14 could then be capture in a "waste" stream while the
other outlet channel(s) can be used to collect the eluted
molecules.
[0039] One of the advantages of the microfluidic system 10 is that
solution exchange happens very quickly (e.g., a couple
milliseconds). This allows one to measure dynamic events as they
occur in the millisecond time scale. If one had to do this with
pipetting or a slow microfluidic method only events in the second
or minute time scale would be accessible. Further, the contents of
the solutions are not the only important factors. One could have
two solutions with different pH or temperatures. These solutions
could be used to elute bound molecules, as mentioned above, test
the response of materials to these conditions, or bring biosamples
into a temperature required for a specific event to occur, like
nucleic acid denaturation, annealing, or amplification.
[0040] FIG. 7 illustrates another embodiment of a microfluidic
system 70. This embodiment includes a two-stage exchange system.
The first stage of the microfluidic system 70 is equivalent to that
described with respect to FIG. 1 and similar features are labeled
with the same reference numbers and will not be described again for
clarity purposes. In this embodiment, the outlet channel 44 that
contains the particles 14 acts as an "input" channel to another
stage of solution exchange. In this regard, the outlet channel 44
intersects with a second transfer channel 72. A third inlet channel
74 is provided through which a second exchange fluid 76 is flowed.
This second exchange fluid 76 may be the same as or different from
the first exchange fluid 30. The third inlet channel 74 is coupled
to a pump 88 or the like that is used to deliver the second
exchange fluid 76. This may include a syringe pump as is shown in
FIG. 7 although other pumping devices may be used. The pump 88
interfaces with an inlet 90 that may have optional filters 80 (like
filters 22) to exclude debris and the like.
[0041] The second transfer channel 72 has a proximal end 71 that
starts at the junction of the outlet channel 44 and the third inlet
channel 74 and a distal end 73 that terminates at third outlet
channel 82 and fourth outlet channel 84. The particles 14 that
enter the second transfer channel 72 from the outlet 44 migrate in
a similar manner as described herein to the second exchange fluid
76. The particles 14 remain therein and travel downstream to the
third outlet channel 82 while non-particulate matter (e.g., debris,
impurities) can then be shunted to the fourth outlet channel 84 and
into chamber 86. In this embodiment, there is double-solution
exchange in a very short period of time. For example, as one
example, the first stage of solution exchange (with exchange fluid
30) may include a wash or clean-up while the second stage of
solution exchange (with second exchange fluid 76) may include a
lysing agent. In this example, the nucleus may then be collected in
one of the downstream collection channels 82, 84. As another
example, the first stage of solution exchange (with exchange fluid
30) may include a wash or clean-up while the second stage of
solution exchange (with second exchange fluid 76) may include a
dye. This embodiment is particularly suited for sample preparation
where multiple steps are used. Not only can the microfluidic device
10, 70 be used for immunohistochemistry or selective lysis, it can
also be used for transfection, fixation, and permeabilization.
[0042] FIG. 8A illustrates another embodiment of a microfluidic
system 90. This system 90 includes a first inlet 92 that is
connected to a first inlet channel 94 and second inlet channel 96
that bifurcate from the common first inlet 92. The first inlet 92
is operatively coupled to a source of particles contained in a
fluid (now shown) that is similar to the other embodiments
described herein. Namely, the first inlet 92 is connected to a pump
(not shown) such as a syringe pump or the like that is configured
to flow particles suspended in solution through the first inlet 92
and into the first and second inlet channels 94, 96. The first
inlet 92 may include filters 98 downstream thereof which may be
structures dimensioned (e.g., posts, fins, or the like) to trap
large debris and aggregates while allowing passage of particles. As
seen in FIG. 8A, the first and second inlet channels 94, 96 are
connected respectively, to optional focusing regions 100, 102 which
may comprise a plurality of microfluidic curves as previously
discussed herein.
[0043] The microfluidic system 90 includes a second inlet 104 that
is connected an inlet channel 106. The second inlet 104 is
operatively coupled to an exchange fluid (not shown) that is
similar to the other embodiments described herein. Namely, the
second inlet 104 is connected to a pump such as a syringe pump or
the like that is configured to exchange fluid through the second
inlet 104 and into the inlet channel 106. The second inlet 104 may
include filters 108 downstream thereof which trap large debris and
aggregates similar to those described with respect to other
filters. The focusing regions 100, 102 intersect with the inlet
channel 106 at the beginning of transfer channel 110. Transfer
channel 110 extends for at least 1 cm and terminates at three
outlet channels 112, 114, 116. There is a central outlet channel
112 that is configured to collect particles in the exchange
solution. The two outer outlet channels 114, 116 are configured to
collect waste solution (e.g., fluid suspending particles). The
central outlet channel 112 may be coupled to a collection chamber
118 or, alternatively, the central outlet channel 112 may continue
onward to additional downstream processing such as an analyzer as
described herein. The two outer outlet channels 114, 116 may be
connected to a common collection chamber 120 as is illustrated in
FIG. 8A.
[0044] FIG. 8B illustrates a cross-sectional view (taken along line
B-B' of FIG. 8A) of the fluid layers created within the transfer
channel 110 of the microfluidic system 90 of FIG. 8A. As seen in
FIG. 8B, there are three distinct layers of fluid created including
a center layer 122 of fluid includes the exchange fluid that is
flowed into the microfluidic system 90 via the second inlet 104 as
well as two outer layers 124, 126. Particles 14 have migrated from
the two outer layers 124, 126 into the center layer 122 to reach an
equilibrium position (X.sub.eq). The two outer layers 124, 126
contain particle suspension fluid, namely, the fluid being
delivered into the microfluidic system 90 via the first inlet 92.
This fluid, along with any debris or other material, is transferred
to the two outer outlet channels 114, 116.
[0045] The microfluidic systems 10, 70, 90 may be manufactured
using processes commonly known to those skilled in the art to make
microfluidic devices. For example, the microfluidic system 10, 70,
90 may be designed using software such as AutoCAD (Autodesk, San
Rafael, Calif., USA). Transparency photomasks for these designs can
be printed at 20,000 dots per inch (CAD/Art Services, Inc., Bandon,
Oreg., USA). Molds for replica molding can then be prepared using
these masks. Negative photoresist, SU-8 50 (MicroChem, Newton,
Mass., USA) is spun on a four (4) inch Silicon wafer at 4,000
rotations per minute. The coated wafer is then soft baked at
65.degree. C. for 5 minutes then 95.degree. C. for 15 minutes. The
wafer is then exposed under near UV at 8.0 mW/cm.sup.2 for 30
seconds. A post-exposure bake of the wafer can be carried out at
65.degree. C. for 2 minutes then at 95.degree. C. for 3.5 minutes.
The unexposed photoresist is then developed in SU-8 Developer
(MicroChem) until an isopropyl alcohol rinse produced no white
film. The height of the resulting features can be characterized by
a surface profiler.
[0046] The width of microchannels immediately before and after the
extensional flow region was 67 .mu.m. The height of the features in
the device was 28 .mu.m. The mold is then taped to the lower plate
of a petri dish with features facing up and an approximately 6 mm
layer of Sylgard 184 Silicone Elastomer (Dow Corning, Midland,
Mich., USA), polydimethylsiloxane (PDMS), mixed 10 parts base to 1
part curing agent, is poured on top. The cast mold is then placed
in a vacuum chamber and the chamber was evacuated for 30 minutes to
remove air from the curing polymer. It was then moved to an oven
set to 65.degree. C. for 3 hours. The devices were cut from the
mold and inlet and outlets were punched into the cured polymer.
They were then placed in a plasma cleaner along with slide glasses
to be activated. After a 30 second exposure to air plasma the
activated surfaces of PDMS and glass were placed in contact to form
permanent covalent bonds between the two materials. While a
PDMS-based construction is described herein it should be understood
that the system and methods disclosed herein are not so limited.
Other microfluidic manufacturing methods may be employed.
[0047] The microfluidic systems 10, 70, 90 discussed herein offer
the ability for high-throughput processing of particles 14 for
solution exchange. For example, the microfluidic systems 10, 70, 90
are able to achieve throughputs in excess of 1,000 particles 14 per
second. Further, various aspects of the different embodiments
described herein may be substituted with one another. As an
example, the downstream analyzer 62 may be used with any of the
embodiments described herein. Thus, while several embodiments have
been described herein it should be appreciated that various aspects
or elements are interchangeable with other separate
embodiments.
[0048] While embodiments have been shown and described, various
modifications may be made without departing from the scope of the
inventive concepts disclosed herein. The invention(s), therefore,
should not be limited, except to the following claims, and their
equivalents.
* * * * *